Plant Physiol. (1986) 80, 1-6
0032-0889/86/80/0001/06/$0 1.00/0
Lectins in Castor Bean Seedlings'
Received for publication April 10, 1985 and in revised form August 29, 1985
SUZANNE M. HARLEY2 AND HARRY BEEVERS*
Biology Department, University ofCalifornia, Santa Cruz, California 95064
ABSTRACT
The amounts of the two lectins (ricin and Ricinus communis agglutinin)
in tissues of castor bean seedlings were followed during germination and
early growth. For measurement, lectins in extracts were separately eluted
from Sepharose columns; an antibody to the agglutinin was also used to
detect the lectins by immunodiffusion. The endosperm of the dry seed
contains 3.5 mg total lectin (5.6% of the total seed protein), which
declines by 50% by day 4 and more rapidly thereafter as the tissue is
completely consumed. The cotyledons of the dry seed also contain lectins
but the amounts are less than 1% of those in the endosperm, and, as in
the endosperm, they are constituents of the albumin fraction of the
isolated protein bodies. No lectins were detected in the green cotyledons
of 10-day seedlings that had been exposed to light from day 5. The
embryonic axes of 2-day seedlings contained very small amounts of lectins
but they were not detectable in the aerial parts of seedlings grown for 3
weeks or in cells from endosperm grown in tissue culture.
The ability of proteinases and glycosidases (isolated from endosperm
of 4-day seedlings) to hydrolyze the lectins was examined. No hydrolysis
of the two lectins was observed, but the subunits, separated by reduction
with 2-mercaptoethanol, were hydrolyzed slowly by a proteinase and
some release of mannose was observed in the presence of the glycosidases.
Ricin was converted to its subunits by cysteine and an enzyme in an
endosperm extract accelerated chain separation by glutathione.
The two castor bean lectins, ricin and RCA1,,3 are glycoproteins
with mol wt of 60,000 and 120,000, respectively (25). Ricin is
composed of two subunits linked by a single disulfide bond, an
A-chain which is a potent inhibitor of protein synthesis by 80S
ribosones, and a carbohydrate binding B-chain (30). RCA,, a
strong hemagglutinin, is a tetramer composed of two A'-chains
and two B'-chains. Each A'-chain is joined to a B'-chain by a
single disulfide bond (31), two such heterodimers being held
together by noncovalent forces (3). Ricin and RCA, are serologically related, although ricin has certain antigenic determinants
that RCA, lacks (34).
The lectins are localized in the protein bodies of the castor
bean endosperm (44, 46). The protein bodies isolated from dry
seeds contain two protein fractions, the albumin proteins of the
'Supported by National Science Foundation Grant PCM 78-19575
from the United States National Science Foundation.
2 Predoctoral fellowship from the National Science Foundation. Present address: Biology Department, University of California, Los Angeles,
CA 90024.
3Abbreviations: RCA,, Ricinus communis agglutinin; PBS, phosphate
buffered saline: Hb, hemoglobin; CPase, carboxypeptidase; LeuNA, Lleucyl-,B-napthylamide; ProNA, L-prolyl-,3-naphthylamide; BANA, a-N-
benzoyl-DL-arginine-,f-naphthylamide; ,@-HexNAc'ase, fl-N-acetylhexosaminidase; a-Man'ase, a-mannosidase.
matrix and the globulin proteins of the crystalloid. The lectins
are components of the matrix proteins (44, 46) and are the only
glycoproteins detectable in the protein bodies (8, 44, 46). Agglutinating activity has been detected in extracts of primary axes
and cotyledons (27). Protein bodies have been observed in electron micrographs of castor bean cotyledons (I. J. Mettler, personal communication), and crystalloids have been isolated from
cotyledons of 2-d-old seedlings (41).
We report here the purification of ricin and RCA, from certain
tissues of castor bean seedlings at various stages of growth using
the technique developed for purifying these lectins from seeds by
Nicolson et al. (25) and attempts to degrade ricin using hydrolases
from the endosperm tissue.
MATERIALS AND METHODS
Plant Materials. Castor bean seeds (Ricinus communis L. cv
Hale) were soaked overnight in running tap water, sown in moist
vermiculite, and germinated in the dark at 3O°C and 85% RH.
The time of planting was taken as d 0. Endosperm tissue was
collected from seedlings at daily intervals. To obtain green cotyledons and leaves, 5-d-old seedlings were selected, the endosperms removed, and the seedlings replanted in vermiculite with
the cotyledons exposed. The plants were placed in a growth
chamber at 30°C and grown with a 12-h light/12-h dark cycle,
I E/m2 s. For comparison, similar seedlings
with light at 110
were returned to the dark. The plants were watered daily with
Hoagland solution. Lyophilized cells from an 18-d suspension
culture, derived from castor bean endosperm, were kindly provided by Dr. R. R. Theimer.
Determination of Total Endosperm Protein. Endosperm tissue
was extracted with 1 N NaOH, using 2 ml per endosperm, by
grinding in a mortar and pestle with sand. The homogenate was
centrifuged at 20,000g for 15 min. The supernatant was decanted
through two layers of Miracloth and the pellet re-extracted with
1 N NaOH. One ml of extract was mixed with 1 ml of 1 N HCI
and 1 ml of 20% (w/v) TCA and held on ice for 5 to 10 min.
The precipitated protein was pelleted and the pellet dissolved in
0.5 N NaOH. An appropriate dilution was assayed for protein
(18), using BSA as the standard. The protein content of other
extracts was assayed in a similar manner.
Quantification of the Lectin Levels. All operations were conducted at 4°C. Twenty seedlings of known age were harvested
and their endosperms extracted with 25 to 40 ml 0.1 M Na
phosphate (pH 7.2), 0.15 M NaCl, 3 mM NaN3 by grinding in a
mortar and pestle with sand. The extract was centrifuged at
20,000g for 15 min, the supernatant decanted through two layers
of Miracloth, and the pellet re-extracted. The combined supernatant was brought to 65% saturation with (NH4)2SO4. A crude
lectin fraction precipitated and was collected by centrifuging at
20,000g for 15 min. The (NH4)2SO4 pellet was suspended in
PBS; 10 mm Na phosphate (pH 7.2), 150 mM NaCl, 3 mM NaN3,
dialyzed against PBS, and loaded onto a Sepharose 4B-200
(Sigma) column equilibrated with PBS. The column was washed
with PBS to remove contaminating proteins. Ricin was eluted
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1986 American Society of Plant Biologists. All rights reserved.
2
HARLEY AND BEEVERS
with 7.5 mM N-acetylgalactosamine in PBS; RCA, was eluted
with 50 mm galactose in PBS (25). The fractions containing ricin
and RCA, were pooled separately and the concentrations of
lectins determined from the A280 extinction coefficients (32). To
estimate the overall effectiveness of the purification and assay
procedure an extract from twenty 4-d endosperms was divided
into two equal portions and 5.0 mg ricin (roughly half of that
already present) was added to one of them. Both portions were
carried separately through the complete procedure and eluted
from Sepharose columns. The sample spiked with ricin was then
found to contain 4.35 mg more ricin than the control. The final
figures for the amount of lectin per endosperm were therefore
corrected for overall losses (13%) during the purification procedure. For assays on cotyledons, 12.5 g cotyledons were extracted
in 36 ml PBS and subjected to the same procedure.
Preparation of the Lectins and their Subunits for Enzymic
Digestion. The purification steps were conducted at 4C. Ricin
and RCA1 were purified from extracts of dry castor bean seeds
by elution from Sepharose 4B-200 with N-acetylgalactosamine
and galactose, respectively (25). The A- and B-chains of ricin
were separated on DEAE-cellulose (Whatman DE-52) as described (29), except that 0.5% (v/v) 2-mercaptoethanol was included in all of the buffers and the B-chain was eluted stepwise
with 0.1 M NaCl. The RCA1 subunits were separated on DEAEcellulose (Whatman DE-52) by the method of Saltvedt (39),
except for the inclusion of 0.5% (v/v) 2-mercaptoethanol in all
of the buffers. The concentrations of ricin, RCAI, and the ricin
A- and B-chains were determined using the A280 extinction
coefficients (32). The concentrations of the RCA1 A- and Bchains were measured by the method of Lowry et al. (18), using
BSA as the standard.
Preparation of Hydrolases from Castor Bean Endosperm. ProNAase, CPase, HB-ase, and a mixture of LeuNAase/
BANAase were prepared as previously described (45). ,BHexNAc'ase and a-Man'ase were prepared as described in (12)
and used in combination.
Hydrolysis of the Lectins. One ml of lectin or subunit, 6 to 8
mg/ml, was mixed with 0.1 ml of hydrolase and incubated at
25C. The three alkaline proteases were used in 0.1 M Na phosphate (pH 7.0), 3 mm NaN3, and the other proteases and the
glycosidases in 0.1 M Na citrate (pH 4.5), 3 mM NaN3. At
intervals of 20 to 25 h, an aliquot of reaction mixture was
removed and the reaction stopped by the addition of 0.5 volume
of 20% (w/v) TCA. The protein that precipitated was pelleted
by centrifugation. Aliquots of the supematant were assayed for
amino acids (16), nonamino sugars (4), or amino sugars (38),
using glycine, mannose, and N-acetylglucosamine, respectively,
as standards.
Preparation of Thiol:Protein Disufide Reductase. All steps
except the heat treatment were conducted at 4C. Endosperm
tissue from 4-d-old castor bean seedlings, 36 g, was extracted
with 70 ml of 0.1 M Na phosphate (pH 7.5), 2 mM EDTA, 3 mM
NaN3 by homogenizing in a blender for 1 min. The homogenate
was centrifuged at 20,000g for 15 min, and the supematant
decanted through two layers of Miracloth. The supernatant, 80
ml, was placed in a 250-ml Erlenmeyer flask, brought to 50C in
a water bath (2.5 min), and held at 50C for 12.5 min. The flask
was cooled to 6C by plunging it into ice (3 min). The cooled
solution was centrifuged (20,000g, 15 min) and the supernatant
dialyzed against 50 mM Na phosphate (pH 7.8), to remove small
metabolites that might interfere with the reductase assay.
Thiol:protein reductase was assayed at 25C by monitoring the
formation of oxidized glutathione, using insulin as the disulfide
containing protein (23).
Extraction of Lectins from other Tissue. For cotyledons and
leaves, from older seedlings, 0.5 to 2 g tissue was ground in 25
ml acetone and centrifuged. The pellet was washed thoroughly
Plant Physiol. Vol. 80, 1986
with acetone, dried, and extracted twice with buffer (2% PVP,
150 mm NaCl, 10 mm DTT, 50 mm Na phosphate, pH 7.3),
using 2 ml of buffer per g of tissue. The extracts were centrifuged
at 16,000g for 15 min. Extracts of dried cells from tissue culture
and fresh roots, hypocotyls, and embryo axes were prepared in a
similar manner.
Immunodiffusion. Ouchterlony double diffusion plates were
prepared on microscope slides, using 3 ml of 1% Noble agar
(Difco) in PBS per slide (24). Galactose, 0.1 M, was included in
the agar to prevent any interaction between the lectins and the
carbohydrate chains of the IgG (14). To avoid any interference
from other serum proteins, IgG was purified from serum on
DEAE-cellulose (40). Antiserum to RCA1 prepared in rabbits
was the kind gift of Dr. L. M. Shannon. The concentration of
the purified IgG which also reacts with ricin was adjusted to 1
mg/ml in PBS. For the double diffusion tests, 10 to 25 Al of
concentrated extract was challenged with 10 ,l of IgG. There
was never a reaction of castor bean extracts with IgG purified
from nonimmune serum.
Protein Body Isolation. Protein bodies were isolated from the
endosperm and cotyledons of dry castor bean seeds using the
nonaqueous glycerol method (44). Endosperms were homogenized in a blender, while the cotyledons were ground in a mortar
and pestle with sand. The albumins were solubilized with 10 mm
Tris-HCl (pH 7.5), and the globulins with 0.1 M Tris-HCl (pH
7.5) 1% SDS.
SDS/PAGE. SDS/PAGE was performed in slab gels according to O'Farrell (28). The gels were fixed in 12% (w/v) TCA for
30 min, followed by 60 min in methanol:water:acetic acid,
9:9:2. Proteins were stained with either Coomassie brilliant blue
R (28) or silver (20). Glycoproteins were stained by the thymol/
sulfuric acid method (37).
Hemagglutination Assay. A 2% suspension of red blood cells
in PBS was incubated with an equal volume (25 Ml) of sample in
PBS in a plastic Petri dish at room temperature (14). After 60
min, the cells were examined for agglutination. Trypsinized,
glutaraldehyde-fixed human type A cells (Sigma) were used.
In Vitro Translation. A poly-U directed in vitro translation
system was prepared using wheat germ and used as previously
reported (1 1). Prior to assaying for protein synthesis, 30 Ml of
ribosomes were incubated at 30C in the presence or absence of
40 Mg of ricin, with either 3.2 mm DTT, 3.2 mM cysteine, or no
reductant. The total volume was 110 Ml. After 30 min, the other
translation components were added, bringing the final volume
100
to 0.4 ml and the ricin concentration to Ig/ml.
The amount
of ["4C]phenylalanine incorporated into TCA precipitable products in 20 min was determined.
RESULTS
Lectin Breakdown in Endosperm Tissue. Figure 1 shows the
changes in protein, dry weight, and fresh weight of the endosperm
during germination and early growth. The amount of protein
per endosperm declines gradually until d 3, after which the rate
of decline increases. The dry weight remains high until d 4, and
then declines rapidly. The fresh weight increases to d 6, especially
from d 3 to 6, but falls as the endosperm becomes visibly
senescent.
The amounts of ricin and RCA1 are essentially equal at all
stages (Fig. 2C). The changes in total lectin content of the
endosperm during the 7-d period are shown in Figure 2. Figure
2C shows that the amount of lectin per endosperm decreases
after d 1. The dry seed contains 3.5 mg of lectin, and approximately 50% of it is still present at d 4. From d 4 there is a more
rapid decline in lectin levels. In the dry seed, the lectins constitute
5.6% of the total seed protein (Fig. 2A). The increase in lectin
per total endosperm protein seen around d 5 (Fig. 2A) is not due
to synthesis ofricin and RCAI, as the lectins decline continuously
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1986 American Society of Plant Biologists. All rights reserved.
LECTINS IN CASTOR BEAN SEEDLINGS
3
0.07
E
c
Z
0
0.06
a0
CL
0
-
0.05
E
0.04
._E
J
EC
e
0
-Jc
0.03
0.02
0
E
ci
.
0
15
'5
0
0
CI
o
.N
021
E
N
J
._
10
10
.C
dry 0 1
seed
2 3 4 5 6 7
on a per endosperm basis (Fig. 2C). The proportion of lectin to
other proteins increases around d 5 because the lectins are
degraded more slowly than the bulk protein. A delay in lectin
degradation, compared to the decline of other storage proteins,
has also been observed by Gifford et al. (8). The amounts of
lectins shown in Figure 2 are those measured after specific elution
of ricin and RCA1 from Sepharose 4B, using the A280 extinction
coefficients (32). Closely similar values for ricin and RCA, at all
stages were obtained when the lectins were measured by the
protein assay of Lowry et al. (18).
Enzymes which May Function in Lectin Breakdown. Since the
lectins comprise over 5% of the endosperm protein and are
broken down during germination, enzymes that could possibly
hydrolyze the lectins were examined. Tully and Beevers (45)
characterized five proteases from the endosperm of germinating
castor beans. The proteases were named for the substrates used
to detect them. ProNAase, LeuNAase, and BANAase have alkaline pH optima, and CPase and Hb-ase have acidic optima.
Ion exchange chromatography can be used to yield fractions
enriched for ProNAase, LeuNAase/BANAase, and Hb-ase, and
CPase activities (45). Ricin, RCAI, and the isolated subunits were
incubated separately with each of the four protease fractions
under optimal pH conditions. No release of amino acids from
intact ricin and RCA, could be detected after 83 h. When the
lectin subunits were incubated under similar conditions only the
Hb-ase was able to release amino acids from the lectin subunits.
The rates of release, shown in Table I, are 4 to 5% that of the
hydrolysis of hemoglobin, the substrate used to detect Hb-ase.
When the lectins and subunits were incubated for 83 h with a
mixture of the three alkaline proteases, no amino acid release
could be detected. When the pH was then lowered to 4.5 with
citric acid and the two acidic proteases added, a more rapid
release of amino acids was seen than when the Hb-ase was used
alone (Table I). Again, only the separated subunits were hydrolyzed, not the intact lectins ricin and RCA1. The activities of the
acidic proteases are highest late in germination (45), when the
most rapid decline in lectin was seen (Fig. 2C).
It was perhaps not unexpected that the castor bean proteases
failed to degrade intact ricin and RCA, since ricin is notorious
for its resistance to proteolytic cleavage (24). It is not hydrolyzed
by trypsin (32), chymotrypsin (32), or pepsin (5). However, the
separated A- and B-chains are digested by trypsin and other
proteases (30, 32). The sensitivity of the subunits to proteolysis
0
>
c:1
0
U-
Seedling age,days
FIG. 1. Changes in the protein content, dry wt, and fresh wt of the
castor beam endosperm during germination.
,
5
ci
0
-J
01
E
Ow
E
0
2.0
0
E
0
2.0
EF
0.
0
1.5
'.5
0
10
C
w
0
0
1.0
1.0
Lii
C
.2_
cE
E
01
0.5
E
0
Dry
Seed
0
1
2
3
4
5
6
7
Seedling Age, Days
FIG. 2. Changes in the amount of lectin (ricin + RCA,) in the castor
bean endosperm during germination. At all times during germination,
the amount of ricin (@) was equal to the amount of RCA, (0) as shown
in (c).
Table I. Release ofAmino Acids during Protease Digestion of the
Subunits of the Castor Bean Lectins
Amino Acid Release
Substrate
Hb-ase + CPase, after
Ricin A-chain
Ricin B-chain
RCA A-chain
RCA B-chain
Hb-ase
LeuNAase, ProNAase, BANAase
nmol/h
17.8
8.9
5.3
9.4
22.6
10.9
7.0
11.5
is probably due to the conformational changes the subunits
undergo upon reduction of the disulfide bond holding them
together (3, 33). There are a number of ways the disulfide bond
linking the A- and B-chains could be reduced in vivo.
Glutathione could reduce disulfide bonds. In vitro, the amount
of glutathione oxidized by reaction with the lectin can be determined by measuring NADPH consumption in the presence of
glutathione reductase. Some reduction occurred in the absence
of extract, but the rate was increased in the presence of enzyme
extract (Table II). Thus, the castor bean endosperm appears to
contain an enzyme reducing lectin at the expense of glutathione.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1986 American Society of Plant Biologists. All rights reserved.
4
HARLEY AND BEEVERS
Table II. Reduction of Castor Bean Lectins by Glutathione
Each assay contained 25 mg/ml lectin, 3.3 mM glutathione, 0.2 mM
NADPH, 0.5 unit glutathione reductase, ±0.1 ml extract of endosperm
tissue of 4-d-old castor bean seedlings.
NADPH Consumed
Lectin
-Extract
+Extract
nmol/min
Ricin
9.32
12.9a
RCA
8.04
20.3a
aThese figures have been corrected for NADPH oxidation in the
absence of added lectin, which was less than 5% of the corrected rates.
Table III. Effect of Reducing Agents on the Ability of Ricin to Inhibit
Protein Synthesis in a Cell-free Translation System Preparedfrom
Wheat Germ
Protein synthesis was determined as the amount of ['4C]phenylalanine
incorporated into protein in 20 min.
Incorporation of [14C]
-Phe(CPM)
Reductant
Inhibition
-Ricin
+Ricin
dpm
%
None
9,886
8,149
18
DTT
9,896
2,076
79
Cysteine
7,120
62
2,724
However, while there is only one interchain disulfide bond, the
B chain of ricin contains four interchain disulfides (31). We do
not know which bonds were reduced in our system. Glutathionerequiring thiol:protein disulfide oxidoreductases have been purified from bovine liver and rat liver and were capable of reducing
the interchain disulfide bond found in ricin (1). A protein disulfide reductase that uses NADPH or NADH has been described
from peas (13). NADPH-dependent thioredoxin reductase from
wheat will reduce insulin or ribonuclease in the presence of
thioredoxin (42).
Cysteine could also reduce the disulfide bond linking the lectin
A- and B-chains. Free ricin A-chain is a more effective inhibitor
of cell-free protein synthesis than the entire ricin molecule (30).
If a reductant such as 2-mercaptoethanol or DTT is included in
the translation system, A-chain is liberated (30). The data in
Table III show that cysteine was effective in generating free Achain. In the absence of reductant, protein synthesis was inhibited
18% by ricin. In the presence of DTT or cysteine, inhibition was
increased 3- to 4-fold due to the release of A-chain.
Because ricin and RCA, are glycoproteins (32), glycosidases
which could hydrolyze the oligosaccharide chains (15) were also
studied. The oligosaccharide chains of the lectins are asparagine
linked and composed of two N-acetylglucosamine residues and
four to seven mannose residues (30). Binding studies with concanavalin A have shown that the mannoses are terminal and/or
2-0-linked (30). The oligosaccharide structures have not been
determined, but from their composition it can be concluded that
they are of the 'simple' or 'high-mannose' type chains, which
contain only N-acetylglucosamine and mannose. As such, the
mannose linkages are probably a and the N-acetylglucosamine
are ft. When ricin, RCA1, or the lectin subunits were incubated
with a mixture of f3-HexNAc'ase and a-Man'ase, an extremely
slow release of nonamino sugars could be detected from the
subunits, but not from the whole lectins. Approximately 0.5
nmol of mannose equivalent was released per h. This was 0. 1 %
of the rate of hydrolysis of p-nitrophenyl-a-mannoside. No release of amino sugars was detected. As with the proteases, the
activities of the glycosidases are highest late in germination (12)
when the most rapid decline in lectin levels was seen (Fig. 2C).
Plant Physiol. Vol. 80, 1986
Lectins in Other Tissues. Protein bodies were isolated from
the cotyledons of dry castor bean seeds, by the nonaqueous
method used to isolate protein bodies from the endosperm (44).
The protein bodies from cotyledons resembled those from endosperm (44) under light microscopy using phase optics. Both
types of protein bodies can be differentially extracted to give
albumin and globulin protein fractions. Figure 3 shows the SDS/
PAGE profile of the albumins and globulins of protein bodies
from cotyledons and endosperm. Differences in the protein
profiles can be seen in the lower mol wt globulins (Fig. 3A). Two
proteins present in the endosperm indicated by dots to the right
of the bands in lane 4 are not observed in the cotyledon and
cotyledons have an albumin band (dot to the right of lane 5) that
is missing from the endosperm. After reduction by 2-mercaptoethanol, the endosperm and the cotyledon albumins each have
a band that the other lacks (Fig. 3B, lanes 3 and 5). Bands which
correspond to ricin and RCA, in the absence of 2-mercaptoethanol can be seen in the albumin proteins from both cotyledons
A
1 2 3 4 5 6 78
B
1 23 45 67 8
~~~~~~~.
-w.
aim~~~~*-
FIG. 3. SDS/PAGE of protein fractions of cotyledon and endosperm
protein bodies from dry castor bean seeds. The gel was a linear gradient
of 6 to 20% acrylamide, stained with Coomassie brilliant blue R. The
proteins in (A) were not reduced with 2-mercaptoethanol, while those in
(B) were reduced. A, lanes 1 and 7, ricin; lanes 2 and 8, RCA1; lane 3,
endosperm albumins; lane 4, endosperm globulins; lane 5, cotyledon
albumins; lane 6, cotyledon globulins. B, lane 1, ricin; lane 2, RCA1; lane
3, endosperm albumins; lane 4, endosperm globulins; lane 5, cotyledon
albumins; lane 6, cotyledons globulins; lanes 7 and 8, low and high mol
wt standards (Bio-Rad), the mol wt from bottom to top being 14,300,
21,000, 30,000. 43,000, 68,000, 94,000, and 130,000.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1986 American Society of Plant Biologists. All rights reserved.
5
LECTINS
and endosperm (Fig. 3). The protein bands which correspond
the lectins (Fig. 3A) and the lectin subunits (Fig. 3B)
also
column. There are species of ricin which do not bind Sepharose (17, 21). Nevertheless, it is quite clear that the endosperm
cells maintained in tissue culture do not contain the high
of lectins present in seed endosperm (Fig. 2).
to
stained
positively for glycoprotein.
level
When an extract of 4-d-old cotyledons was
pharose 4B, a protein peak was detectable
separated
when
0.1 M
on
lactose
used to elute the column (Fig. 4). When
examined by SDS/PAGE, bands which corresponded
and RCA1 were detected. Furthermore, the proteins
lactose formed precipitin bands when challenged
fusion tests with anti-RCA, IgG, but not IgG purified
nonimmune serum. The fractions eluted by
agglutinated red blood cells. Therefore, the cotyledons
seeds contain both ricin and RCA1, but the
than those found in the endosperm of the
seedling (Table
was
this
DISCUSSION
material
to
eluted
in
lactose
are
castor
much
same
IV).
When 5-d-old seedlings
are exposed to light, Chl synthesis
within 1 d and as the cotyledons become photosynthetic
organs the levels of ricin and RCA, drop. The immunoreaction
begins
to antibody RCA,
was
barely detectable
of greening cotyledons
at 9 d and
tests were still positive using
seedlings kept
in the dark.
in concentrated
absent
extracts
of
from
Double diffusion
for extracts of cotyledons and leaves from
or leaves from castor bean plants grown
greenhouse conditions.
Embryo axes from 2-d-old
seedlings
were
3-week-old
for
4
the amounts of seed lectins
plants
organs of
grown
serologically
or
lectins
and
some of
from
related molecules in
these
seeds
have been
measured by immunoassays. Legumes such as Phaseolus vulgaris
(2), Psophocarpus tetragonolobus (u9), Glycine max (36), and
have been examined for distribution of
Dolichos biflorus
seed lectins and their fate during germination and early growth.
The
of the
has been especially useful
for studying lectins in the Gramineae such as wheat, where the
(43)
sensitivity
immunoassays
agglutinain is present in very low amounts (1 gg/seed compared
lectin
to 1-4 mg/seed in legumes [22]). These plants contain
one
or
collection
a
isolectins.
of
extracts
by d 10. Ouchterlony
cotyledons
tests
many plant species contain specific
by
double
also
of the
levels
The seeds of
114-d
negative
seedlings
months
Castor
bean, the object of the present study, has
ricin and
which although
RCA1,
sharing
minants, are quite distinct. Thus,
the
amounts
of each
lectin in
many
it is not
an
two
lectins,
antigenic
deter-
to determine
possible
extract or mixture
with the
antibody to RCA1. However, the two lectins can be purified and
the basis of their different carboseparated from each other
hydrate binding specificities (25) in stepwise elution from Seon
contain
low
amounts
ricin and RCA,. A 10-ml Sepharose column
ricin and RCA, from 1.2 g of embryo
2-d seedlings.
Protein bands that coincided with ricin
RCA,
PAGE were isolated from hypocotyls of
seedlings.
15 g of tissue gave 1 ml of concentrate which required
sensitive silver stain (20) for visualization
0.1
analyzed by SDS/PAGE. Extracts of
4-d
were devoid of Sepharose binding material
gave negative
was used
to
purify
axes from
and
on
4-d
SDS/
However,
the
when
roots
from
seedlings
pharose 4B.
castor bean
The
1.78 mg of RCA1.
lectins
are
seedlings
mg of
seed contains 1.73 mg of ricin and
During germination
depleted simultaneously
the levels
RCAl.
have
declined
and early growth the two
and in the endosperm
to
of 74
0.053 mg ricin and 0.056
In the endosperm of the dry seed both lectins are present
the protein bodies, where they form part of the water
matrix proteins (44, 46). Protein bodies are also present
in
soluble
in
the
and
Ouchterlony tests.
An extract of 1.5 g dry weight of endosperm
grown
tissue culture contained a small amount
material, but no detectable ricin or RCA, could
Sepharose 4B with lactose. The same weight
germinating seeds would contain 10.2 mg
6.2 mg at d 7 (Fig. 2), amounts that would give large
noprecipitate and easily purified on a small (20-40 ml) Sepharose
part of the albumin
much less lectins than the endo-
cotyledons, where again ricin and RCA1
fraction.
Cotyledons contain
cells
of
immunoreactive
be
eluted
of
of lectin
(Table IV) and as the
content drops to undetectable
d-old seedlings
sperm
low
amounts
at
were
are
cotyledons turn green
levels. The embryo
the lectin
axes
from 2-
and hypocotyls from 4-d-old seedlings contain
of lectin. From each of these tissues the lectins
purified using the technique developed
by Nicolson
et
aL
a
(25).
example,
and root
I
0
~.5k-
trast,
root
lectins from Arachis hypogaea (35),
the
j
0
II
260
230
30
a
hypocotyl
Vigna radiata (9). In confound in pods, stems and
stems and derived callus from
of G. max (6) and Pisum sativum (7)
roots,
Psophocarpus (39),roots
are not identical to the seed lectins.
During germination and early growth
0.02
and
a-galactosidase from
immunoreactive materials
leaves of Dolichos (43)
0.
0C
a
and shown
hemagglutinin
~~~~~~0.04-
I
CS
4c
legume tissues have been purified by the
to be identical to the seed lectins; for
lectin from Onobrychis vicitfolia (10) hypocotyl
Some lectins from
same technique
of castor bean the lectins
in the
endosperm are degraded most rapidly after the bulk of the
(Fig. 3; 8). This is in contrast
storage protein has been
to
where the lectins of the
(43) and Glycine
cotyledons are mobilized simultaneously with the other storage
reserves. The small amounts of lectins in the young cotyledons
depleted
FIG. 4. Purification of ricin and RCA,
cotyledons of germinating castor beans by Sepharoseaffinity
raphy.
from
4-d-old
an
chromatog-
Dolichos
(36)
of castor bean disappear
Table IV. Lectin Quantities in the Cotyledons
4-d-old Castor
Bean
and
Endosperm of
E:C
Cotyledon, %
of Endosperm
mg
lectin/g fresh wt
mg lectin/g dry wt
mg lectin/seedling
2.4
0.13
6.8
0.24
1.7
0.005
.8.5
28.3
as
the seedling becomes established and
in the organs of most young
found
decline
germination (43).
50%
highest levels in actively growing regions (22).
In an attempt to elucidate the breakdown of the lectins in the
substrates for proteases and
endosperm, they were tested
carbohydrases isolated from the tissue, where they are contained
in the vacuole f(26).None of the enzymes
able to break down
as
340
lectin, % of total
protein
5.4
Lectins
to undetectable levels within 2 to 3 weeks of
However, 344d-old seedlings of wheat contain
of the agglutinin present in ungerminated grains, with the
seedlings
Seedlings
Endosperm Cotyledon
photosynthetic.
was
4.1
0.15
27.3
the native lectins, but
some release of amino acids
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1986 American Society of Plant Biologists. All rights reserved.
was observed
6
HARLEY AND BEEVERS
when the SH-proteinase Hbase was added to the separated A and
B chains of both ricin and RCA,. Evidence was obtained that
glutathione could reduce the S-S bonds of the lectins to bring
about chain separation and the reaction was stimulated by an
enzyme in the endosperm extract. Cysteine also brought about
chain separation. A slow release of mannose from the subunits
was observed when the glycosidases were added. From what is
known in other systems, it seems likely that both protein and
carbohydrate moieties would be more readily hydrolyzed if they
were first cleaved by a peptide:N-glycosidase (15), but no such
activity could be detected with crude extracts or the carbohydrases isolated from the endosperm tissue ( 12).
Acknowledgments-We thank Dr. L. M. Shannon, University of CaliforniaRiverside for his gift of antiserum to RCA,, and Dr. R. R. Theimer, Botanisches
Institut Munchen for supplying dried cells of endosperm from suspension culture.
LITERATURE CITED
1. BARBIERI L, MG BATTELLI, F STIRPE 1982 Reduction of ricin and other plant
toxins by thiol:protein disulfide oxidoreductases. Arch Biochem Biophys
216: 380-383
2. BORREBAECK CAK, B MATTIASSON 1983 Distribution of a lectin in tissues of
Phaseolus vulgaris. Physiol Plant 58: 29-32
3. CAWLEY DB, LL HOUSTON 1979 Effect of sulfhydryl reagents and protease
inhibitors on sodium dodecyl sulfate-heat induced dissociation of Ricinus
communis agglutinain. Biochim Biophys Acta 81: 51-62
4. DuBoIs M, KA GILLES, JK HAMILTON, PA REBERS, F SMITH 1956 Colorimetric
method for determination of sugars and related substances. Anal Chem 28:
350-356
5. FUNATSU G, M FUNATSU 1977 Limited hydrolysis of ricin D with alkaline
protease from Bacillus subtilis. Agric Biol Chem 41: 1309-1310
6. GADE W, MA JACK, JB DAHL, EL SCHMIDT, F WOLD 1981 The isolation and
characterization of a root lectin from soybean (Glycine max (L.), cultivar
Chippewa). J Biol Chem 256: 12905-12910
7. GATEHOUSE JA, D BOULTER 1980 Isolation and properties of a lectin from the
roots of Pisum sativun (garden pea). Physiol Plant 49: 437-442
8. GIFFORD DJ, JS GREENWOOD, JD BEWLEY 1983 Vacuolation and storage
protein breakdown in the castor bean endosperm following inhibition. J Exp
Bot 34: 1433-1443
9. HAAss D, R FREY, M THIESEN, H KAUss 1981 Partial purification of a
hemagglutinin associated with cell walls from hypocotyls of Vigna radiata.
Planta 151: 490-496
10. HAPNER KD, JE ROBBINS 1979 Isolation and properties of a lectin from
sainfoin (Onobrychis viciifolia, Scop.). Biochim Biophys Acta 580: 186-197
11. HARLEY SM, H BEEVERS 1982 Ricin inhibition of in vitro protein synthesis by
plant ribosomes. Proc Natl Acad Sci USA 79: 5935-5938
12. HARLEY SM, H BEEVERS 1985 Characterization and partial purification of
three glycosidases from castor bean endosperm. Phytochemistry. 24:
1459-1464
13. HATCH MD, JF TURNER 1960 A protein disulphide reductase from pea seeds.
Biochem J 76: 556-562
14. HOWARD J, J KINDINGER, LM SHANNON 1979 Conservation of antigenic
determinants among different seed lectins. Arch Biochem Biophys 192:457465
15. KOBATA A 1979 Use of endo- and exoglycosidases for structural studies of
glycoconjugates. Anal Biochem 100: 1-14
16. LEE YP, T TAKAHASHI 1966 An improved colorimetric determination of amino
acids with the use if ninhydrin. Anal Biochem 14: 71-77
17. LIN TTS, SSL Li 1980 Purification and physiochemical properties of ricins
and agglutinins from Ricinus communis. Eur J Biochem 105: 453-459
18. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275
19. MEIMETH T, K TRAN THANH VAN, J-L MARCOTTE, TH TRINH, AE CLARKE
1982 Distribution of lectins in tissues, derived callus, and roots of Psophocarpus tetragonolobus (winged bean). Plant Physiol 70: 579-584
Plant Physiol. Vol. 80, 1986
20. MERRIL CR, D GOLDMAN, SA SEDMAN, MH EBERT 1981 Ultrasensitive stain
for proteins in polyacrylamide gels shows regional variation in cerebrospinal
fluid proteins. Science 21: 1437-1438
21. MISE T, G. FUNATSU, M ISHIGURO, M FUNATSU 1977 Isolation and characterization of ricin E from castor beans. Agric Biol Chem 41: 2041-2046
22. MISHKIND M, K KEEGSTRA, BA PALEVITA 1980 Distribution of wheat germ
agglutinin in young wheat plants. Plant Physiol 66: 950-955
23. MORIN JE, DF CARMICHAEL, JE DIXON 1978 Characterization, kinetics and
comparative properties of thiol:protein disulfide oxidoreductase. Arch
Biochem Biophys 189: 354-363
24. MUNOZ J 1971 Double diffusion in plates. In CA Williams, MW Chase, eds,
Methods in Immunology and Immunochemistry, Vol 3. Academic Press,
New York, pp 146-160
25. NICOLSON GL, J BLAUSTEIN, ME ETZLER 1974 Characterization of two plant
lectins from Ricinus communis and their quantitative interactions with a
murine lymphoma. Biochemistry 13: 196-204
26. NISHIMURA M 1982 pH in vacuoles isolated from castor bean endosperm.
Plant Physiol 70: 742-744
27. NsiMBA-LuBAKI M, WJ PEUMANS, AR CARLIER 1983 Isolation and characterization of a lectin from Euphorbia heterophylla seeds. Biochem J 215: 141 145
28. O'FARRELL PH 1975 High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007-4021
29. OLSNES S, A PIHL 1973 Different biological properties of the two constituent
peptide chains of ricin, a toxic protein inhibiting protein synthesis. Biochemistry 12: 3121-3126
30. OLsNEs S, A PHIL 1976 Abrin, ricin, and their associated agglutinins. In P
Cuatrecases, ed, The Specificity and Action of Animal, Bacterial and Plant
Toxins. Chapman and Hall, London, pp 129-173
31. OLSNES S, A PIHL 1982 Toxic lectins and related proteins. In P Cohen, S van
Heyningen, eds, Molecular Action of Toxins and Viruses. Elsevier Biomedical Press, Amsterdam, pp 51-105
32. OLSNES S, K REFSNES, TB CHRISTENSEN, A PIHL 1975 Studies on the structure
and properties of the lectins from Abrus precatorius apd Ricinus communis.
Biochim Biophys Acta 405: 1-10
33. OLSNEs S, E SALTVEDT 1975 Conformation-dependent antigenic determinants
in the toxic lectin ricin. J Immunol 1 14: 1743-1748
34. PAPPENHEIMER AM, S OLSNEs, AA HARPER 1974 Lectins from Abrus precatorius and Ricinus communis. I. Immunochemical relationships between
toxins and agglutinins. J Immunol 113: 835-841
35. PUEPPKE SG 1979 Distribution of lectins in the Jumbo Virgirwia and Spanish
varieties of peanuts, Arachis hypogaea L. Plant Physiol 64: 575-580
36. PUEPPKE SG, WD BAUER, K KEEGSTRA, AL FERGUSON 1978 Role of lectins
in plant-microorganism interactions. II Distribution of soybean lectin in
tissue of Glycine max (L.) Merr. Plant Physiol 61: 779-784
37. RACUSEN D 1979 Glycoprotein detection in polyacrylamide gel with thymol
and sulfuric acid. Anal Biochem 99: 474-476
38. ROULEAU M 1980 Effects of cations, sugars, detergents, sulfhydryl compounds,
and cryoprotective agents on the colorimetric determination of N-acetylglucosamine by the method of Reissig. Anal Biochem 103: 144-151
39. SALTVEDT E 1976 Structure and toxicity of pure ricinus agglutinin. Biochim
Biophys Acta 451: 536-548
40. SHANNON LM, SE MILLS 1976 Purification by immunoadsorbtion chromatography ofthe normal and a mutant form of the B2 subunit of Escherichia coli
tryptophan synthase. Eur J Biochem 63: 563-568
41. SOBOLEV AM 1966 On the state of phytin in the aleurone grains of mature and
germinating seeds. Soviet Plant Physiol 13: 177-183
42. SUSKE G, W WAGNER, H FOLLMAN 1979 NADPH-dependent thioredoxin
reductase and a new thioredoxin from wheat. Z Naturforsch 34c: 214-221
43. TALBOTCF, ME ETZLER 1978 Develoment and distribution ofDolichos biflorus
lectin as measured by radioimmunoassay. Plant Physiol 61: 847-850
44. TULLY RE, H BEEVERS 1976 Protein bodies of castor bean endosperm. Isolation, fractionation, and the characteristics of protein components. Plant
Physi.il 58: 710-716
45. TULLY RE, H BEEVERS 1978 Proteases and peptidases of castor bean endosperm. Enzyme characterization and changes during development. Plant
Physiol 62: 746-750
46. YOULE RJ, AHC HUANG 1976 Protein bodies from the endosperm of castor
beans. Subfractionation, protein components, lectins, and changes during
germination. Plant Physiol 58: 703-709
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1986 American Society of Plant Biologists. All rights reserved.